Herpes viruses have been suggested as potential vectors for gene delivery. This could, for example, either be to the nervous system or elsewhere in the body for gene therapy, vaccine or other purposes, or to cells in culture or to animal models of disease. However, while HSV has a number of potential advantages as a vector in that it can infect a wide variety of cell types in vitro and in vivo and can accept large DNA insertions allowing the delivery of multiple genes, infection of most cell types with HSV will result in lytic replication or other toxic effects of the virus. Thus for use as a vector HSV must usually be disabled in some way to prevent or minimise these effects.
HSV can be disabled in a number of ways, and this includes when used as a helper virus in the growth of so-called amplicon vectors which consist of plasmids containing herpes origin and packaging sequences which can be replicated in the presence of a helper virus after transfection into permissive cells. For example genes required for replication in all cell types can be inactivated, which must be complemented for growth in culture, these including one or other or both of the essential immediate early genes ICP4 or ICP27. Alternatively genes required for pathogenesis in vivo but which are not required for growth in culture can be removed, such as ICP34.5 or ICP6, as can further genes the deletion of which reduces toxicity further. These can for example include the other IE genes ICPO, ICP22, and ICP47, the inactivation of ICPO and/or ICP22 reducing the efficiency of virus replication in culture unless these are also complemented in the cell line used for virus growth. The production of effective and practical vector viruses therefore depends on a balance of appropriately minimised toxicity in the target cell type, and on the ability to grow the virus in culture, in some cases requiring the use of a cell line complementing at least some of the inactivating mutations in the virus. As a general rule, the greater the number of mutations in the virus, the harder the virus will be to grow in culture. HSV vectors generally are reviewed in Coffin and Latchman, 1996.
It can be seen from the above that particularly attractive genes for inactivation in the production of HSV vectors are one or more of the five IE genes, as inactivation of these will for ICPO, ICP4, ICP22 or ICP27 at least, also reduce levels of other proteins the expression of which is stimulated by these IE gene products. However if these genes are inactivated replication in culture will either be blocked (ICP4 and ICP27) or reduced (ICPO and ICP22), and thus for efficient replication the inactivated genes must be complemented in the cell line used for virus growth.
However an alternative means by which the levels of functional IE proteins can be reduced, rather than by including inactivating mutations in the IE genes themselves, is to include an inactivating mutation in the gene encoding VP 16 (Ace et al., 1988). VP 16 is a virion protein that together with cellular factors is responsible for the trans-activation of HSV IE gene promoters after infection. Thus inclusion of specific inactivating mutations in VP 16 results in a virus in which IE gene expression is reduced, although not blocked completely (Ace et al., 1989). This may be advantageous in the production of an HSV vector virus as inactivation of a function in one gene (VP16) results in reduced levels of expression of multiple IE genes.
The gene for VP 16 cannot however be deleted from the virus as it is also an essential structural protein. Specific mutations are therefore used which reduce or abolish the transactivating activity of VP 16, but still allow the protein to fulfill its structural function (Ace et al., 1988). Viruses including this type of mutation—specifically insertion of a linker sequence into the gene for VP 16 as in virus mutant in1814 (Ace et al., 1989)—are essentially avirulent in vivo, giving reduced growth both in vivo and in culture (Ace et al., 1989). Growth of stocks of viruses including such a mutation is thus of reduced efficiency as compared to viruses lacking the mutation. The mutation in VP16 can be partially compensated for by the inclusion of HMBA in the media (MacFarlane et al., 1992), but cell lines cannot be used that have been engineered to express an unaltered copy of VP 16 without the generation of virus in the culture in which the mutation has been repaired. This is because, as the gene cannot be deleted from the virus due to its essential structural role, the inclusion of an unaltered copy of the gene for VP 16 in the cell line used for virus growth would result in the generation of virus containing the unaltered VP 16 sequence by homologous recombination between the mutated VP16 sequence in the virus and the unaltered VP16 sequence in the cell line. Moreover complementation of the VP16 mutation by such a cell line would in any case result in the production of new virions containing fully functional VP16, which when used as a vector in non-complementing cells would activate IE gene expression, exactly as the mutation in VP16 was intended to reduce.
The problem therefore remains of how to efficiently grow stocks of HSV including mutations in the gene for VP16 which affect the trans-activating properties of the protein, such that the mutation in the virus cannot be repaired during virus growth.